Effect of miR-513a-5p on etoposide-stimulating B7-H1 expression in retinoblastoma cells

J Huazhong Univ Sci Technolog Med Sci. 2012 Aug;32(4):601-606. doi: 10.1007/s11596-012-1004-8. Epub 2012 Aug 11.

Abstract

This study investigated the effect of etoposide, an anticancer chemotherapy drug, on B7-H1 expression in retinoblastoma (Rb) cells and the role of miR-513a-5p in the process. Rb cells were divided into control and etoposide groups. In the etoposide group, cells were treated with etoposide at different concentrations (2.5, 5, 10, 20 and 40 μg/mL) for 24 h. Those given no treatment of etopside served as controls. Reverse transcription polymerase chain reaction (RT-PCR), fluorescence quantitative PCR and flow cytometry were performed to measure the mRNA and protein expression of B7-H1 in Rb cells. The mRNA expression of miR-513a-5p in Rb cells before and after etoposide treatment was also detected by fluorescence quantitative PCR. The miR-513a-5p mimics and the miR-513a-5p inhibitor were transfected into Rb cells separately, and fluorescence quantitative PCR and flow cytometry were used to detect the effect of the miR-513a-5p mimics or inhibitor on B7-H1 expression. TargetScan5.2 was employed to predict the miR-513a-5p binding sites in the 3'-untranslated region of B7-H1 mRNA. Luciferase reporter plasmids carrying this site were prepared and transfected into Rb cells and luciferase activity analyzed. The results showed that etoposide stimulated the mRNA and protein expression of B7-H1 in Rb cells, which reached a maximal level after treatment with 5 μg/mL etoposide (P<0.05). However, miR-513a-5p expression was decreased in Rb cells after etoposide treatment. When the miR-513a-5p inhibitor was added, B7-H1 expression was increased with the concentration of the miR-513a-5p inhibitor (P<0.05). Moreover, B7-H1 expression was decreased gradually with the concentration of the miR-513a-5p mimics increased (P<0.01). Additionally, the miR-513a-5p mimics were found to inhibit the luciferase activity. It was concluded that etoposide can promote B7-H1 expression in Rb cells, which may be associated with chemoresistance. The promoting effect of etoposide on B7-H1 expression can be reversed by miR-513a-5p mimics. MiR-513a-5p inhibits the mRNA and protein expression of B7-H1 via binding to the 3'-UTR of B7-H1 mRNA.

MeSH terms

  • B7-H1 Antigen / genetics*
  • Cell Line, Tumor
  • Etoposide / pharmacology*
  • Gene Expression / drug effects*
  • Gene Expression / genetics
  • Humans
  • MicroRNAs / genetics*
  • Retinoblastoma / genetics*

Substances

  • B7-H1 Antigen
  • CD274 protein, human
  • MIRN513 microRNA, human
  • MicroRNAs
  • Etoposide